Integration of parallel pathways for flight control in a hawkmoth reflects prevalence and relevance of natural visual cues

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field, elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a prioritization for generating behavior that supports hawkmoth safety rather than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Reviewer #2 (Public review):

Summary

Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight. The authors linked their behavioral results to visual scene statistics in the hawkmoths' natural environment. The partition of ventral and dorsal visuomotor pathways is well in line with differences in visual cue frequencies. The response hierarchy, however, seems to be dominated by dorsal features, that are less frequent, but presumably highly relevant for the animals' flight safety.

Strengths

The data are very interesting and unique. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

Weaknesses

While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

I find the majority of the data, which are also the data supporting the main claims of the paper, compelling. However, the measurements of flight height are less solid than the rest and I think these data should be interpreted more carefully.

Reviewer #3 (Public review):

The authors have significantly improved the paper in revising to make its contributions distinct from their prior paper. They have also responded to my concerns about quantification and parameter dependency of the integration conclusion. While I think there is still more that could be done in this capacity, especially in terms of the temporal statistics and quantification of the conflict responses, they have a made a case for the conclusions as stated. The paper still stands as an important paper with solid evidence a bit limited by these concerns.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a priortiziation for generating behavior that supports hawkmoth safety rather than than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Weaknesses:

The work would be further clarified and strengthened by additional explanation included in the main text, figure legends, and methods that would permit the reader to draw their own conclusions more feasibly. It would be helpful to have all figure panels referenced in the text and referenced in order, as they are currently not. In addition, it seems that sometimes the incorrect figure panel is referenced in the text, Figure S2 is mislabeled with D-E instead of A-C and Table S1 is not referenced in the main text at all. Table S1 is extremely important for understanding the figures in the main text and eliminating acronyms here would support reader comprehension, especially as there is no legend provided for Table S1. For example, a reader that does not specialize in vision may not know that OF stands for optic flow. Further detail in figure legends would also support the reader in drawing their own conclusions. For example, dashed red lines in Figures 3 and 4 A and B are not described and the letters representing statistical significance could be further explained either in the figure legend or materials to help the reader draw their own conclusions.

We appreciate the suggestions to improve the clarity of the manuscript. We have extensively re-structured the entire manuscript. Among others, we have referenced all figure panels in the text in the order they appear. To do so, we combined the optic flow and contrast measurements of our setup with the methods description of the behavioural experiments (formerly Figs. 5 and 2, respectively). This new figure 2 now introduces the methods of the study, while the remainder of Fig. 2, which presented the experiments that investigated the vetrolateral and dorsal response in more detail, is now a separate figure (Fig. 3). This arrangement also balances the amount of information contained in each figure better.

Reviewer #2 (Public review):

Summary:

Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight.

Strengths:

The data are very interesting, unique, and compelling. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

Weaknesses:

While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

We thank the reviewer for the feedback, and the suggestions for improvement of the manuscript (our implementations are detailed below). We fully agree that this study raises several intriguing questions regarding the dorsal visual response, including how the animals perceive and respond to rotational optic flow in their dorsal visual field, particularly since rotational optic flow may be processed separately from translational optic flow.

In our free-flight setup, it was not possible to generate rotational optic flow in a controlled manner. To explore this aspect more systematically, a tethered-flight setup would be ideal, or alternatively, a free-flight setup integrated with virtual reality. This would be a compelling direction for a follow-up study.

Reviewer #3 (Public review):

The central goal of this paper as I understand it is to extract the "integration hierarchy" of stimulus in the dorsal and ventrolateral visual fields. The segregation of these responses is different from what is thought to occur in bees and flies and was established in the authors' prior work. Showing how the stimuli combine and are prioritized goes beyond the authors' prior conclusions that separated the response into two visual regions. The data presented do indeed support the hierarchy reported in Figure 5 and that is a nice summary of the authors' work. The moths respond to combinations of dorsal and lateral cues in a mixed way but also seem to strongly prioritize avoiding dorsal optic flow which the authors interpret as a closed and potentially dangerous ecological context for these animals. The authors use clever combinations of stimuli to put cues into conflict to reveal the response hierarchy.

My most significant concern is that this hierarchy of stimulus responses might be limited to the specific parameters chosen in this study. Presumably, there are parameters of these stimuli that modulate the response (spatial frequency, different amounts of optic flow, contrast, color, etc). While I agree that the hierarchy in Figure 5 is consistent for the particular stimuli given, this may not extend to other parameter combinations of the same cues. For example, as the contrast of the dorsal stimuli is reduced, the inequality may shift. This does not preclude the authors' conclusions but it does mean that they may not generalize, even within this species. For example, other cue conflict studies have quantified the responses to ranges of the parameters (e.g. frequency) and shown that one cue might be prioritized or up-weighted in one frequency band but not in others. I could imagine ecological signatures of dorsal clutter and translational positioning cues could depend on the dynamic range of the optic flow, or even having spatial-temporal frequency-dependent integration independent of net optic flow.

We absolutely agree that in principle, an observed integration hierarchy is only valid for the stimuli tested. Yet, we do believe that we provide good evidence that our key observations are robust also for related stimuli to the ones tested:

Most importantly, we found that both pathways act in parallel (and are not mutually exclusive, or winner-takes-all, for example), when the animals can enact the locomotion induced by the dorsal and ventrolateral pathway. We tested this with the same dorsal cue (the line switching direction), but different behavioural paradigms (centring vs unilateral avoidance), and different ventrolateral stimuli (red gratings of one spatial frequency, and 100% nominal contrast black-and-white checkerboard stimuli which comprised a range of spatial frequencies) – and found the same integration strategy.

Certainly, if the contrast of the visual cues was reduced to the point that the dorsal or ventrolateral responses became weaker, we would expect this to be visible in the combined responses, with the respective reduction in response strength for either pathway, to the same degree as they would be reduced when stimuli were shown independently in the dorsal and ventrolateral visual field.

For testing whether the animals would show a weighting of responses when it was not possible to enact locomotion to both pathways, we felt it was important to use similar external stimuli to be able to compare the responses. So we can confidently interpret their responses in terms of integration. Indeed, how this is translated to responses in the two pathways depends a) on the spatiotemporal tuning, contrast sensitivity and exact receptive fields of the two systems, b) the geometry of the setup and stimulus coverage, and therefore the ability of the animals to enact responses to both pathways independently and c) on the integration weights.

It would indeed be fascinating to obtain this tuning and the receptive fields, and having these, test a large array of combinations of stimuli and presentation geometries, so that one could extract integration weights for different presentation scenarios from the resulting flight responses in a future study.

We also expanded the respective discussion section to reflect these points: l. 391-417. We also updated the former Fig. 5, now Fig. 6 to reflect this discussion.

The second part of this concern is that there seems to be a missed opportunity to quantify the integration, especially when the optic flow magnitude is already calculated. The discussion even highlights that an advantage of the conflict paradigm is that the weights of the integration hierarchy can be compared. But these weights, which I would interpret as stimulus-responses gains, are not reported. What is the ratio of moth response to optic flow in the different regions? When the moth balances responses in the dorsal and ventrolateral region, is it a simple weighted average of the two? When it prioritizes one over the other is the response gain unchanged? This plays into the first concern because such gain responses could strongly depend on the specific stimulus parameters rather than being constant.

Indeed, we set up stimuli that are comparable, as they are all in the visual domain, and since we can calculate their external optic flow and contrast magnitudes, to control for imbalances in stimulus presentation, which is important for the interpretation of the resulting data.

As we discussed above, we are confident that we are observing general principles of the integration of the two parallel pathways. However, we refrained from calculating integration weights, because these might be misleading for several reasons:

(1) In situations where the animals can enact responses to both pathways, we show that they do so at the full original magnitudes. So there are no “weights” of the hierarchy in this case.

(2) Only when responses to both systems are not possible in parallel, do we see a hierarchy. However, combined with point (1), this hierarchy likely depends on the geometry of the moths’ environment: it will be more pronounced the less both systems can be enacted in parallel.

(3) The hierarchy also does not affect all features of the dorsal or ventrolateral pathway equally. The hawkmoths still regulate their perpendicular distance to ventral gratings with dorsal gratings present, to same degree as with only ventral grating - because perpendicular distance regulation is not a feature of the dorsal response. And while the hawkmoths show a significant reduction in their position adjustment to dorsal contrast when it is in conflict with lateral gratings (Fig. 4C), they show exactly the same amount of lateral movement and speed adjustment as for dorsal gratings alone, when not combined with lateral ones (Fig. 4D and Fig. S3A). So even for one particular setup geometry and stimulus combination, there clearly is not one integration weight for all features of the responses.

We extended the discussion section to clarify these points “The benefit of our study system is that the same cues activate different control pathways in different regions of the visual field, so that the resulting behaviour can directly be interpreted in terms of integration weights” (l. 448-451)

l. 391-417, we also updated the former Fig. 5, now Fig. 6 to reflect this discussion.

The authors do explain the choice of specific stimuli in the context of their very nice natural scene analysis in Fig. 1 and there is an excellent discussion of the ecological context for the behaviors. However, I struggled to directly map the results from the natural scenes to the conclusions of the paper. How do they directly inform the methods and conclusions for the laboratory experiments? Most important is the discussion in the middle paragraph of page 12, which suggests a relationship with Figure 1B, but seems provocative but lacking a quantification with respect to the laboratory stimuli.

We show that contrast cues and translational optic flow are not homogeneously distributed in the natural environments of hawkmoths. This directly related to our laboratory findings, when it comes to responses to these stimuli in different parts of their visual field. In order to interpret the results of these behavioural experiments with respect to the visual stimuli, we did perform measurements of translational optic flow and contrast cues in the laboratory setup. As a result, we make several predictions about the animals’ use of translational optic flow and contrast cues in natural settings:

a) Hawkmoths in the lab responded strongest to ventral optic flow, even though it was not stronger in magnitude, given our measurements, than lateral optic flow. Thus, we propose that the stronger response to ventral optic flow might be an evolutionary adaptation to the natural distribution of translational optic flow cues.

b) In the natural habitats of hawkmoths, dorsal coverage is much less frequent that ventrolateral structures generating translational optic flow, yet the hawkmoths responded with a much higher weight to the former. Moreover, in our flight tunnel experiments, the animals responded with the same or higher weights to dorsal cues, which had a lower magnitude of translational optic flow and contrast than the same cues in the ventrolateral visual field. So we showed, combining behavioural experiments and stimulus measurements in the lab that the weighting of dorsal and ventrolateral cues did not follow their stimulus magnitude in the lab. Moreover, comparing to the natural cue distributions, we suggest that the integration weights also did not evolve to match the prevalence of these cues in natural habitats.

We integrated the measurements of natural visual scene statistics in the new Fig. 6, to relate the behavioural findings to the natural context also in the figure structure, and sequence logic of the text, as they are discussed here.

The central conclusion of the first section of the results is that there are likely two different pathways mediating the dorsal and the ventrolateral response. This seems reasonable given the data, however, this was also the message that I got from the authors' prior paper (ref 11). There are certainly more comparisons being done here than in that paper and it is perfectly reasonable to reinforce the conclusion from that study but I think what is new about these results needs to be highlighted in this section and differentiated from prior results. Perhaps one way to help would be to be more explicit with the open hypotheses that remain from that prior paper.

We appreciate the suggestion to highlight more clearly what the open questions that are addressed in this study are. As a result, we have entirely restructured the introduction, added sections to the discussion and fundamentally changed the graphical result summary in Fig. 6, to reflect the following new findings (and differences to the previous paper):

The previous paper demonstrated that there are two different pathways in hummingbird hawkmoths that mediate visual flight guidance, and newly described one of them, the dorsal response. This established flight guidance in hummingbird hawkmoths as a model for the questions asked in the current study, which are very different in nature from the previous paper.

The main question addressed in the current study is how these two flight guidance pathways interact to generate consistent behaviour? Throughout the literature of parallel sensory and motor pathways guiding behaviour, there are different solutions – from winner-takes-all to equal mixed responses. We tested this fundamental question using the hummingbird hawkmoth flight guidance systems as a model.

This is the main question addressed in the various conflict experiments in this study, and we show that indeed, the two systems operate in parallel. As long as the animals can enact both dorsal and optic-flow responses, they do so at the original strengths of the responses. Only when this is not possible, hierarchies become visible. We carefully measured the optic flow and contrast cues generated by the different stimuli to ensure that the hierarchies we observed were not generated by imbalances of the external stimuli.

- Does the interaction hierarchy of the two pathways follow the statistics of natural environments? We did show qualitatively previously how optic flow and contrast cues are distributed across the visual field in natural habitats of the hummingbird hawkmoth. In this study, we quantitatively analysed the natural image data, including a new analysis for the contrast edges, and statistically compared the results across conditions. This quantitative analysis supported the previous qualitative assessment that the prevalence of translational optic flow was highest in the ventral and lowest in the dorsal visual field in all natural habitat types. The distribution of contrast edges across the visual field did depend on habitat type much stronger than visible in the qualitative analysis in the previous paper. When compared to the magnitude of the behavioural responses, and considering that the hummingbird hawkmoth is predominantly found in open and semi-open habitats, the natural distributions of optic flow and contrast edges did not align with the response hierarchy observed in our laboratory experiments. Dorsal cues elicited much stronger responses relative to ventrolateral optic flow responses than would be expected.

To provide a more complete picture of the dorsal pathway, which will be important to understand its nature, and also compare to other species, we conducted additional experiments that were specifically set up to test for response features known from the translational optic flow response. To compare and contrast the two systems. These experiments here allowed us to show that the dorsal response is not simply a translational optic flow reduction response that creates much stronger output than the ventrolateral optic flow response. We particularly show that the dorsal response was lacking the perpendicular distance regulation of the optic flow response, while it did provide alignment with prominent contrasts (possibly to reduce the perceived translational optic flow), which is not observed in the ventrolateral optic flow response. The strong avoidance of any dorsal contrast cues, not just those inducing translational optic flow, is another feature not found in the ventrolateral pathway.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Many comparisons between visual conditions are made and it was confusing at times to know which conditions the authors were comparing. Thinking of a way to label each condition with a letter or number so that the authors could specify which conditions are specifically being compared would greatly enhance comprehension and readability.

We appreciate this concern. To be able to refer to the individual stimulus conditions in the analysis and results description, we gave each stimulus a unique identifier (see table S1), and provided these identifiers in the respective figures and throughout the text. We hope that this makes the identification of the individual stimuli easier.

Consider adding in descriptive words to the y-axis labels for the position graphs that would help the reader quickly understand what a positive or negative value means with respect to the visual condition.

We did now change the viewpoint on the example tracks in Figs. 2-5, to take a virtual viewpoint from the top, not as the camera recorded from below, which requires some mental rotation to reconcile the left and right sides. Moreover, we noticed that the example track axes were labelled in mm, while the axes for the plots showing median position in the tunnel were labelled in cm. We reconciled the units as well. This will make it easier to see the direct equivalent of the axis (as well as positive and negative values) in the example tracks in those figures, and the median positions, as well as the cross-index.

There are no line numbers provided so it is a bit challenging to provide feedback on specific sentences but there are a handful of typos in the manuscript, a few examples:

(1) Cue conflict section, first paragraph: "When both cues were presented to in combination, ..." (remove to)

(2) The ecological relevance section, first paragraph, first sentence: "would is not to fly"

(3) Figure S3 legend: explanation for C is labeled as B and B is not included with A

We apologise for the missing line numbers. We added these and resolved the issues 1-3.

Reviewer #2 (Recommendations for the authors):

- The pictograms in Fig. 1a were at first glance not clear to me, maybe adding l, r, d, v to the first pictogram could make the figure more immediately accessible.

We added these labels to make it more accessible.

- I would suggest noting in the main text that the red patterns were chosen for technical reasons (see Methods), if this is correct.

We added this information and a reference to the methods in the main text (lines 100-102).

- "Thus, hawkmoths are currently the only insect species for which a partitioning of the visual field has been demonstrated in terms of optic-flow-based flight control [33-35]." I think that is a bit too strong and maybe it would be more interesting to connect the current data to connected data in other insects to perhaps discuss important similarities. Ref 32 for example shows that fruit flies weigh ventral translational optic flow considerably more than dorsal translational optic flow. Reichardt 1983 (Naturwissenschaften) showed that stripe fixation in large flies (a behaviour relying in part on the motion pathway) is confined to the ventral visual field, etc...

We have changed this sentence to acknowledge partitioning in other insects, and motivating the use of our model species for this study: While fruit flies weight ventral translational optic flow stronger than dorsal optic flow, the most extreme partitioning of the visual field in terms of optic-flow-based flight control has been observed in hawkmoths [33-35]. (lines 60-62)

- I think the statistical differences group mean differences could be described in more detail at least in Fig. 2 (to me the description was not immediately clear, in particular with the double letters).

We added an explanation of the letter nomenclature to all respective figure legends:

Black letters show statistically significant differences in group means or median, depending on the normality of the test residuals (see Methods, confidence level: 5%). The red letters represent statistically significant differences in group variance from pairwise Brown–Forsythe tests (significance level 5%). Conditions with different letters were significantly different from each other. The white boxplots depict the median and 25% to 75% range, the whiskers represent the data exceeding the box by more than 1.5 interquartile ranges, and the violin plots indicate the distribution of the individual data points shown in black.

- "When translational optic flow was presented laterally" I would use a more wordy description, since it is the hawkmoth that is controlling the optic flow and in addition to translational optic flow, there might also be rotational components, retinal expansion etc.

We extended the description to explain that the moths were generating the optic flow percept based on stationary gratings in different orientations, by way of their flight through the tunnel. Lines 127-129

- While it is clearly stated that the measure of the perpendicular distance from the ventral and dorsal pattern via the size of the insect as seen by the camera is indirect, I would suggest to determine the measurement uncertainty of distance estimate.

- Connected to above - is the hawkmoth area averaged over the entire flight and is the variance across frames similar in all the stimuli conditions? Is it, in principle, conceivable that the hawkmoths' pitch (up or down) is different across conditions, e.g. with moths rising and falling more frequently in a certain condition, which could influence the area in addition to distance?

There are a number of sources that generate variance in the distance estimate (which was based on the size of the moth in each video frame, after background subtraction): the size of the animal, the contrast with which the animal was filmed (which also depended on the type of pattern in the tunnel – it was lower with ventral or dorsal patterns as a background than with lateral ones), and the speed of the animal, as motion blur could impact the moth’s image on the video. The latter is hard to calibrate, but the uncertainty related to animal size and pattern types could theoretically be estimated. However, since we moved between finishing the data acquisition for this study and publishing the paper, the original setup has been dismantled. We could attempt to recreate it as faithfully as possible, but would be worried to introduce further noise. We therefore decided to not attempt to characterise the uncertainty, to not give a false impression of quantifiability of this measure. For the purpose of this study, it will have to remain a qualitative, rather than a quantitative measure. If we should use a similar measure again, we will make sure to quantify all sources of uncertainty that we have access to.

The variance in area is different between conditions. Most likely, the animals vary their flight height different for different dorsal and ventral patterns, as they vary their lateral flight straightness with different lateral visual input. For the reasons mentioned above, we cannot disentangle the effects of variations in flight height and other sources of uncertainty relating to animal size in the video frames. We therefore averaged the extracted area across the entire flight, to obtain a coarse measure of their flight height. Future studies focusing specifically on the vertical component or filming in 3D will be required to determine the exact amount of vertical flight variation.

- Results second paragraph, suggestion: pattern wavelength or spatial frequency instead of spatial resolution.

- Same paragraph, suggestion: For an optimal wavelength/spatial frequency of XX

We corrected these to spatial frequency.

- Above Fig 3- "this strongly suggests a different visual pathway". In my opinion it would be better to say sensory-motor /visuomotor pathway or to more clearly define visual pathway? Could one in principle imagine a uniform set of local motion sensitive neurons across the entire visual field that connect differentially to descending/motor neurons.

We appreciate this point and changed this, and further instances in the manuscript to visuomotor pathway.

- If I understood correctly, you calculated the magnitude of optic flow in the different tunnel conditions based on the image of a fisheye camera moving centrally in the tunnel, equidistant from all walls. I did not understand why the magnitude of optic flow should differ between the four quadrants showing the same squarewave patterns. Apologies if I missed something, but maybe it is worth explaining this in more detail in the manuscript.

We recognize that this point may not have been immediately clear and have therefore provided additional clarification in the Methods and results section (lines 106-111, 543-549). We anticipated differences in the magnitude of optic flow due to potential contrast variations arising from the way the stimuli were generated—being mounted on the inner surfaces of different tunnel walls while the light source was positioned above. On the dorsal wall, light from the overhead lamps passed through the red material. For laterally mounted patterns, the animals perceived mainly reflected light, as these tunnel walls were not transparent.

A similar principle applied to the background, which consisted of a white diffuser allowing light to pass through dorsally, but white non-transmissive paper laterally, with a 5% contrast random checkerboard patterns. The ventral side presented a more complex scenario, as it needed to be partially transparent for the ventrally mounted camera. Consequently, the animals perceived a combination of light reflections from the red patterns and the white gauze covering the ventral tunnel side, against the much darker background of the surrounding room.

To ensure that the observed flight responses were not artifacts of deviations in visual stimulation from an ideal homogeneous environment, we used the camera to quantify the magnitude of optic flow and contrast patterns under these real experimental conditions. This approach also allowed us to directly relate the optic flow measurements taken indoors to those recorded outdoors, as we employed the same camera and analytical procedures for both datasets.

Reviewer #3 (Recommendations for the authors):

In addition to the considerations above I had a few minor points:

There are so many different directions of stimuli and response that it is quite challenging to parse the results. Can this be made a little easier for the reader?

We appreciate this concern. To be able to refer to the individual stimulus conditions in the analysis and results description, we gave each stimulus a unique identifier (see table S1), and provided these identifiers in the respective figures and throughout the text. We hope that this makes the identification of the individual stimuli easier.

One suggestion (only a suggestion): I found myself continuously rotating the violin plots in my head so that the lateral position axis lined up with the lateral position of the tunnel icons below. Consider if rotating the plots 90 degs would help interpretability. It was challenging to keep track of which side was side.

We did discuss this with a number of test-readers, and tried multiple configurations. They all have advantages and drawbacks, but we decided that the current configuration for the majority of testers was the current one. To help the mental transformations from the example flight tracks in the figures, we now present the example flight tracks in Figs. 2-5 in the same reference frame as the figures showing median position (so positive and negative values on those axes correspond directly), and changed the view from a below the tunnel to an above the tunnel view, as this is the more typical depiction. We hope that this enhances readability.

Are height measurements sensitive to the roll and pitch of the animal? I suspect this is likely small but worth acknowledging.

They are indeed. These effects are likely small but contribute to the overall inaccuracy, which we could not quantify in this particular setup (see also response to reviewer 2 on that point), which is why the height measurements have to be considered a qualitative approximation rather than a quantification of flight height. We added text to acknowledge the effects of roll and pitch specifically (lines 657-658)

The Brown-Forsythe test was reported as paired but this seems odd because the same moths were not used in each condition. Maybe the authors meant something different by "paired" than a paired statistical design?

Indeed, the data was not paired in the sense that we could attribute individual datapoints to individual moths across conditions. We applied the Brown-Forsythe test in a pairwise manner, comparing the variance of each condition with another one in pairs each, to test if the variance in position differed across conditions. We did phrase this misleadingly, and have corrected it to „The variance in the median lateral position (in other words, the spread of the median flight position) was statistically compared between the groups using the pairwise Brown–Forsythe tests“ l. 187-188

There is some concern about individual moth preferences and bias due to repeated measures. I appreciate that the individual moth's identity was not likely known in most cases, but can the authors provide an approximate breakdown of how many individual moths provided the N sample trajectories?

This is a very valid concern, and indeed one we did investigate in a previous study with this setup. We confirmed that the majority of animals (70%, 68% and 53% out of 40 hawkmoths, measured on three consecutive days) crossed the tunnel within a randomly picked window of 3h (Stöckl et al. 2019). We now state this explicitly in the methods section (lines 594-597). Thus, for the sample sizes in our study, statistically, each moth would have contributed a small number of tracks compared to the overall number of tracks sampled.

The statistics section of the methods said that both Tukey-Kramer (post-hoc corrected means) and Kruskal-Wallis (non-parametric medians) were done. It is sometimes not clear which test was done for which figure, and where the Kruskal-Wallis test was done there does not seem to be a corrected statistical significance threshold for the many multiple comparisons (Fig. 2). It is quite possible I am just missing the details and they need to be clarified. I think there also needs to be a correction for the Brown-Forsythe tests but I don't know this method well.

We first performed an ANOVA, and if the test residuals were not normally distributed, we used a Kruskal-Wallis test instead. For the post-hoc tests of both we used Tukey-Kramer to correct for multiple comparisons. The figure legends did indeed miss this information. We added it to clarify our statistical analysis strategy and refer to the methods section for more details (i.e. l. 185-186). All statistical results, including the type of statistical test used, have been uploaded to the data repository as well.

The connection to stimulus reliability in the discussion seems to conflate reliability with prevalence or magnitude.

We have rephrased the respective discussion sections to clearly separate the prevalence and magnitude of stimuli, which was measured, from an implied or hypothesized reliability (lines 510-511).

Line numbers would be helpful for future review.

We apologize for missing the line numbers and have added them to the revised manuscript.